FOLIA HISTOCHEMICA
ET CYTOBIOLOGICA
Vol. 45, Supp. 1, 2007
pp. 87-91
Pronuclear scoring as a predictor of embryo quality in
in vitro fertilization program
Magdalena Depa-Martynow, Piotr Jedrzejczak, Leszek Pawelczyk
Division of Infertility and Reproductive Endocrinology, Department of Gynecology and Obstetrics,
Karol Marcinkowski University of Medical Sciences in Poznañ, Poland
Abstract: Many strategies have been proposed for the selection of viable embryos for transfer in human assisted reproduction. These have included morphological scoring criteria for 20, 28, 44 and 68 h after insemination. The embryo selection
is based on morphology, degree of fragmentation and development to the 8-cell. All have shown some correlation with
implantation. However, the overall success of these methods is still limited, with over 50% of all transferred embryos failing to implant. Pronuclear zygote morphology has gained much attention recently due to its positive value in predicting
implantation and pregnancy. This prospective study involved 178 conventional IVF patients only. The key aspects of pronuclear scoring and namely the presence of a cytoplasmic halo were related to day 3 of development and morphology in a retrospective study. The Z-score and the presence/absence of a halo had significant effect on the rate of development on day 3
embryo. Low Z-score result in slow development and poor morphology. The absence of a halo also resulted in slow and
poor development, low morphology, increased fragmentation.
Key words: Pronuclear scoring - Embryo quality - Assisted reproduction
Introduction
Precise selection of embryos and prediction of
implantation is probably the most pressing issue in
assisted reproduction. Various embryo scoring system have been described to assess the developmental
potential of human day 2 or 3 preimplantation
embryos. In the most commonly used systems the
blastomeres cleavage rate (i.e. number of blastomeres), the shape and size of the blastomeres and
the amount of anucleated fragments are estimated.
Several studies have shown that regardless of minor
fragmentation, the optimal cleavage rate would be the
most important criteria when selecting embryos for
transfer [1, 2]. Variation in zona thickness, embryo
symmetry and the presence of multinucleated blastomeres has also been shown to affect implantation
rates [1, 3, 4].
In recent years, there has been growing interest in
the assessment of pronuclear morphology to select
Correspondence: Magdalena Depa-Martynow, Division
of Infertility and Reproductive Endocrinology, Department
of Gynecology and Obstetrics, University of Medical Science,
33 Polna St., 60-535 Poznañ, Poland; tel.: (+4861) 8419604,
fax: (+4861) 8419612, e-mail: [email protected]
the most viable and competent embryos. In this
regard in vitro fertilization (IVF) human pronucleate
zygotes are scored on the basis of pronuclear alignment, size, number, equality and distribution of
nucleoli, cytoplasmic heterogenity and presence or
absence of cytoplasmic halos [5, 6, 7, 8]. This scoring system correlated positively with the implantation and delivery rates in pronuclear stage transfers.
Many different pronuclear scoring systems have been
proposed to select high- quality embryos. Unfortunately, there is no standard zygote grading system
used thought assisted reproduction laboratories. The
same holds true for other systems of evaluating
embryo morphology. As a result, comparisons of the
systems used correlations of embryo quality with
success rates between different laboratories are
vague. Two main systems for assessing pronuclear
morphology were developed by Scott and Smith
(1998) and Tessarik et al. (2000) [5, 9]. A pronuclear
scoring system, as well as other embryo development
markers and patient status, may be useful in determining the number and quality of embryos to transfer. The early scoring parameters currently used in
clinical settings have a sound biological basis that
reflects on the physiological process communication
of the gametes and zygote forming.
86
M. Depa-Martynów et al.
Materials and methods
A total of 787 zygotes from 178 IVF cycles were investigated in
the Division of Infertility and Endocrinology of Reproduction in
Poznañ, between September 2005 and December 2006. All patient
enrolled in this study were female factor of infertility and were
treated by conventional IVF.
For ovarian stimulation 150-300 IU human menopausal
gonadotrophin (Menopur, Ferring) injections were started daily
beginning from day 3 and after verification of pituitary suppression
with the gonadotrophin - releasing hormone agonist triptorelin
(Decapeptyl 0.1 mg/day, Ferring). Triptorelin injections were started daily on day 21 of the menstrual cycle preceding the treatment
cycle.
Final oocyte maturation was triggered with 10 000 IU of
human chorionic gonadotrophin (HCG, Pregnyl) when the leading
two follicles were >17 mm or the leading cohort measured 15-16
mm in diameter. Transvaginal ultrasound guided oocyte retrieval
was performed under conscious sedation at 35-36h post - HCG.
Oocytes were collected directly in to warmed (37°C) G-MOPS
Plus medium (Vitrolife, Sweden) and placed in CO2 incubator
(Heracell, Heraeus).
Men's ejaculate after liquefaction, were analyzed for sperm
density, motility and morphology. Spermatozoa were purified by
centrifugation through discontinuous SpermGrad (Vitrolife), and
prepared with swim - up method.
For fertilization a conventional IVF procedure were used. All
retrieved oocyte - corona-cumulus -complexes were placed in 4-well
dishes with G-FERT Plus medium (Vitrolife, Sweden) and inseminated 100 000 - 300 000 motile sperm after 3 h. from oocyte
retrieval. At about 16-18h after insemination, oocytes were examinated for the presence of pronuclei and polar bodies. Fertilization
considered normal when two clearly distinct pronuclei were present.
The presence or absence of a cytoplasmic halo. About 16-18h
after fertilization a differential cytoplasmic distribution for pronuclear embryos was reported. According to the appearance (presence) of a halo a halo-positive zygote or a halo-negative zygote
were classified (Fig. 1).
The Z-Score. The zygotes were scored according to the Z-scoring
system [10]. The system took account of nuclear size and alignment and nucleoli (nucleolar precursor bodies, NPB) number and
distribution.
Briefly, Z-1 zygotes had equal numbers of NPB aligned at the
pronuclear junction. The absolute number was not counted but was
between three and six. Z-2 zygotes had equal number and sizes of
nucleoli (between three and six) which were equally scattered in
the two nuclei. Z-3 zygotes had equal numbers of NBP of equal
sizes in the same nuclei but with one nucleus having alignment at
the pronuclear junction and the other with scattered nucleoli.
Zygotes with unequal numbers (a difference of more than two
nucleus) and /or sizes of nucleoli were also considered as Z-3. Z-4
zygotes were those with pronuclei that were separated, of very different sizes or periphery located (Fig. 2).
Day 3 embryo scoring. The morphology of an embryo was noted
68h (day3) after insemination. The embryos grade A-D according
to degree of cytoplasm fragmentation and the number of blastomeres was scored. Grade A contained the best embryos: at least
7 blastomeres (7-9 blastomeres) and maximum 20% of cytoplasm
fragmentation. Grade B embryos have 7-9 cells also but with over
20% of cytoplasmic fragmentation. Grade C has 4-6 cells embryos
with maximum 20% fragmented cytoplasm and finally grade D,
contained the worst (morphologically lower) embryos with 4-6
cells and over 20% of fragmentation (Fig. 3).
Statistical analysis. Results were expressed as percentages. Statistical analysis of the data was performed using Excel program and
Fig. 1. The presence or absence od a cytoplasmic halo: Halo-positive zygote (A); Halo-negative zygote (B).
Fig. 2. S-score: Z1 zygote (A); Z2 zygote (B); Z3 zygote (C); Z4
zygote (D).
Student's test of the Statistica 5.0 software. The comparisons were
made by using the χ2-test with continuity correction.
Results
A total of 787 pronuclear oocytes were obtained in our
laboratory over a 16-month period. Each pronucleated
oocyte was assessed individually for:
1. The presence or absence of a cytoplasmic halo,
2. Z-scoring.
Each of these two morphological parameters of
zygote was correlated with grades of embryos: A, B,
C. D.
The presence or absence of a cytoplasmic halo
In all, 787 pronuclear oocytes an accurate scoring of
presence or absence of a cytoplasmic halo was
checked. A total of 628 (79.8 %) zygotes was included in group of zygotes with halo positive effect and
159 (20.2%) in group of zygotes without cytoplasmic
halo. The comparison of a grade A, B, C and D day 3
embryos and presence or absence of a cytoplasmic
halo was evaluate.
87
Predictor of embryo quality
Table 1. Percentages of embryos grade A, B, C and D obtained
from zygotes with the presence or absence of a cytoplasmic halo.
In all, 787 pronuclear oocytes an accurate scoring of presence or
absence of a cytoplasmic halo was checked. A total of 628 (79.8%)
zygotes was included in group of zygotes with halo positive effect
and 159 (20.2%) in group of zygotes without cytoplasmic halo.
The comparison of a grade A, B, C and D day 3 embryos and presence or absence of a cytoplasmic halo was evaluate.
The difference between these two groups was statistically different;
aP, bP, cP, dP represents statistical significance for quality of embryo
acquired from zygotes with presence and absence of a cytoplasmic halo.
Figure 3. Embryos grade: grade A (A); grade B (B); grade C (C);
grade D (D).
tistically different (P<0.001), (P<0.05), (P<0.001),
(P<0.001) respectively. The percentages of embryo
development from halo positive and halo negative
zygotes are shown in Table 1.
Z- scoring
Figure 4. Percentages of embryo grades A, B, C and D obtained
from zygote type Z1, Z2, Z3 and Z4. All embryos were scored for
day 3 morphology regardless of Z-score. The score for each Zscore group was then analyzed. There was a significant difference
in the day 3 morphology embryos between the four Z-groups.
From Z1 and Z2 there was an increase in grades A and B embryos
respectively. The Z3 and Z4 pronuclear embryos had poor day 3
morphology mostly (grade C and D embryos)
* - represents statistical significance for quality of embryo,
P value <0,001
** - represents statistical significance for quality of embryo,
P value <0,0001
***- represents statistical significance for quality of embryo,
P value <0,00001
During the study period 358 (57.0%) grade A
embryos, 191 (30.4%) grade B embryos, 79 (12.6%)
grade C embryos and none (0%) grade D embryos
derived from halo positive zygotes was observed.
From a total of 159 halo negative zygotes 3 (1.9%)
grade A embryos, 37 (23.0%) grade B embryos, 52
(32.7%) grade C embryos and 67 (42.1%) grade D
embryos we obtained. The difference between these
two groups for grade A, B, C, and D embryos was sta-
The day 3 embryos rates showed statistical differences between embryos formed from zygote types
Z1, Z2, Z3 and Z4. The number of zygotes reached
grade A embryos stage was greater in group Z1
(71,3%) than in group Z2 (22.2%), Z3 (6.5%) and Z4
(0%) (P<0.001), (P<0.0001), (P<0.00001) respectively. Statistical differences among groups were
observed in grade B embryos in four types of
zygotes. The percentage of grade B embryos was the
highest in Z2 group (53.3%). The number of grade B
embryos in Z1, Z3 and Z4 zygote group was significant lower (P<0.001), (P<0.0001), (P<0.0001)
respectively. The presence grade C embryos was also
observed. The most grade C embryos developed
from Z3 zygotes. The statistical differences were
observed between Z3 zygote group and Z1 zygote
group (P<0.0001), Z2 zygote group (P<0.0001), and
Z4 zygote group (P<0.001). The percentage of grade
D embryos was significantly higher in Z4 zygotes
group than in Z1 zygotes group (P<0.00001), Z2
zygotes group (P<0.0001) and Z3 zygotes group
(P<0.001) (Fig. 4)
Discussion
Assessment of development and implantation potential of embryos is a great importance for selection of
embryos for transfer, so as to achieve high pregnancy rates associated with a low number of multiple
pregnancies. For that purpose, scoring of zygotes
88
and cleavage stage embryos has been proposed to
identify and to select embryos for fresh transfer [7,11].
The finding of a positively correlation of appearance of a cytoplasmic halo and embryo quality is in
accordance with a previous study [7,12,13]. The halo
effect has been variously attributed to cytoplasmic
rotation or movement or to the differential distribution
of mitochondria [14].
Payne et al. (1997) were the first to report a subplasmalemmal zone of translucent cytoplasm immediately prior to formation of the male and female pronucleus [14]. Subsequently, this focal clearing within the
cortical cytoplasm (cytoplasmic flare) often progress
to involve the entire cytocortex (halo). This phenomenon is thought to be the manifestation of a microtubule
- organized translocation of mitochondria and other
cytoplasmic components to the centre of the oocyte,
since virtually no detectable mitochondria were found
in the cortical region of fertilized oocytes. More likely,
mitochondria concentrate in the perinuclear cytoplasm
in an ellipsoidal mass [15]. The physiological role of
mitochondrial redistribution in human zygotes is
unknown but it may be speculated that clustering of
mitochondria to perinuclear regions may be involved
in cell cycle regulation by means of calcium mobilization and ATP liberation [15-19]. In addition, location
of mitochondria next to the pronuclei would allow
immature mitochondria, as seen in zygotes, to complete maturation presuming that some input from the
nucleus is needed [17, 20].
Another parameter evaluated in zygote morphology
is the appearance of a nucleolar precursor bodies
(NPB) during pronucleus formation. The data presented in this paper indicate that, the morphology of the
pronuclear oocyte has a direct effect on continued in
vitro development, namely increased quality of day 3
embryos, giving a greater chance for successful
implantation and pregnancy.
Previous studies have also clearly demonstrated the
effect of this parameter on developing embryo [7,12,
21-24]. Since the oocyte is ovulated with all the
machinery in place for these initial cell cycles, it is
unlikely that nuclear events will have much of an
effect [25]. The one aspects: equal number of NPB
between the nuclei is a necessary event in any mitotic
cell. Unequal number of NPB results in abnormal cell
cycles, which may be the foundation of cancer cells
[12]. The asynchrony in the numbers and pattern of
NPB between the nuclei leads also to the lack of polarization [15]. The coalescence and reformation of functional nucleoli has profound effect on the ability of
cells to grow and function [23].
Embryos with equal number of NBP, Z1 and Z2, have
normal development and good morphology. The lack of
polarization of the NPB in the Z3 and Z4 embryos is a
morphological indication of a lack of chromatin polar-
M. Depa-Martynów et al.
ization, which will lead to abnormal, slower and much
more poor development as recorded in this data.
Generally cell cycle events are controlled by checkpoints, or surveillance systems, that either block or
allow cell cycle transitions. Incomplete, incorrect or
damaged transitions or events are stopped by these
mechanisms [26]. Some of these surveillance mechanisms operate to ensure that chromosome duplication
and completion occur correctly, and that chromosome
alignment on the spindle is both timely and spatially
correct. If incorrect alignment is detected, a signal is
transduced /initiated, which stops the system until the
correct alignment is attained [27]. This could account
for the slower development, more fragmentation and
abnormal cleavage and finally fewer embryos continuing development to the 3 day stage [25]. When transferred, they result in fewer implantation [5, 10, 28-31].
The present study and the results of other published
studies confirm that when there exists polarization of
NPB in both pronuclei and the pronuclei are similar
size (Z1) and additionally a halo is observed, embryo
quality and development is improved. The pronuclear
morphology scoring is an additional criterion for
selecting embryos for extended culture. The Z-score
and halo score in combination with day 3 embryo morphology is useful in determining the most suitable
number of embryos for transfer, and achieving the
optimal chance of conception while reducing the risk
of high order multiple pregnancy.
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